Screening saikosaponin d (SSd)-producing endophytic fungi from Bupleurum scorzonerifolium Willd

Saikosaponin d (SSd) is an important bioactive compound of traditional Chinese medicinal plant Bupleurum scorzonerifolium Willd. and exhibits many effects, such as anti-tumor, anti-inflammation and immunomodulatory. Since endophytic fungi possess the natural capacity to produce the similar secondary metabolite to that of their host plants, they are promising as alternative sources of plant bioactive natural products. In this study, in order to search for SSd-producing strains, endophytes were isolated from B. scorzonerifolium and were authenticated by the ITS sequence and the translation elongation factor-1alpha gene (TEF-1α) sequence analysis. The profile of metabolites present in the crude exacts was carried out by ultra performance liquid chromatography time-of-flight mass spectrometry (UPLC/Q-TOF-MS) analysis. The results showed that two strains, CHS2 and CHS3 from B. scorzonerifolium could produce SSd by UPLC/Q-TOF-MS analysis, and the amount of SSd produced by strain CHS2 and CHS3 were about 2.17 and 2.40 µg/mL, respectively. CHS2 and CHS3 showed a close phylogenetic relationship to Fusarium oxysporum and Fusarium acuminatum, respectively. According to our concern, no endophytic fungi capable of producing SSd from B. scorzonerifolium have been found before. Our clear intention was to isolate and identify these endophytic fungi that produce important active secondary metabolites, and then study the strains that produce this compound on a large scale through fermentation or even genetic study, to provide a feasible and more convenient way for the production of SSd. Supplementary Information The online version contains supplementary material available at 10.1007/s11274-022-03434-x.


Introduction
Epiphytic and endophytic are group of microorganisms that live in healthy host plants (Santamaría and Bayman 2005). Endophytes generally exist in various tissues and organs inside the host plants as least a part of their life cycle without directly causing any obvious external diseases and adverse results (Stone et al. 2000;Quilliam and Jones 2010). It is known that endophytes are beneficial to hosts by protecting plants against a variety of biotic and abiotic stresses (Yan et al. 2019;Abeer et al. 2016;Bian et al. 2021). Endophytic fungi possess the natural capacity to produce and accumulate secondary metabolites. These secondary metabolites have various biological activities and can be directly or indirectly used as medicine to treat different diseases (Aly et al. 2010;Nisa et al. 2015;Staniek et al. 2008). In recent years, the metabolites from endophytic microorganisms have shown that endophytes have been valued as potential sources of anti-cancer, antioxidant, 1 3 242 Page 2 of 8 antibiotics and immunosuppressive bioactive compounds (Kamel et al. 2020). Endophytic fungi are regarded as alternative resources of medicinal plants because they can produce the same bioactive compounds as their host plant, such as alkaloids, terpenoids, steroids and phenols.
Nan-Chai-Hu, also named Radix Bupleuri, the root of Bupleurum scorzonerifolium Willd. (Umbelliferae), is a traditional Chinese herb which is used in treating of tumor, allergy and inflammation in Asia (Ashour et al. 2009;The Pharmacopoeia Commission of the PRC 2015). This plant's secondary metabolites might offer us with a variety of active compounds containing flavonoids, essential oils, coumarins, lignans, triterpene saponins, polyacetylenes and alkaloids. These bioactive components are crucial in disease treatments (Ashour and Wink 2011). Among them, saikosaponins (SSs) were found to be the most active compounds, especially saikosaponin d (SSd), which exhibits many effects, such as anti-tumor, anti-inflammation and immunomodulatory (Zhou et al. 2021;Li et al. 2018). In our previous work, the endophytic fungi from B. scorzonerifolium were isolated and the distribution characteristics of endophytes in B. scorzonerifolium have been investigated (Gao et al. 2012).
The central goal of this study was to screen the endophytic fungi with the capability of SSd producing from B. scorzonerifolium. Ultra performance liquid chromatography time-of-flight mass spectrometry (UPLC/Q-TOF-MS) analysis was performed to detect whether the endophytes isolated from B. scorzonerifolium could produce the similar compounds to host plants. Up to now, no endophytic fungi of SSd producing from B. scorzonerifolium have been reported.

Isolation of endophytes from B. scorzonerifolium
Endophytic fungi were isolated from different parts (roots, stems, leaves and flowers) of B. scorzonerifolium with aseptic operation. For surface sterilization, each part of the plant was washed with sterile water, followed by soaking in 75% ethanol (v/v) for 3 min, rinsing twice with sterile water, soaking in 0.1% mercuric chloride (v/v) for 5 min, and rinsing with sterile water for three times. 5 × 5 mm of segments cut from the treated plant material were cultured on potato dextrose agar (PDA) medium (1 L PDA medium containing potato 200 g, dextrose 20 g and agar 12 g) at 25 °C, checking the growth of endophytic fungi in plant segments every day.

Ethanol extraction from endophytic fungi
The endophytic fungi were cultured in potato dextrose broth (PDB) medium (1 L PDB medium containing potato 200 g and dextrose 20 g) at 28 °C and shaking at 160 rpm for 10 days in the dark. The cultured fungi were collected by vacuum filtration, and then grinded into powder in liquid nitrogen. Ethanol extraction from the fungal powder with 70% ethanol was evaporated with a rotary evaporator (Eyela, Jpn). The extractions were prepared for SSd analysis by UPLC/Q-TOF-MS.
The sample is analyzed by Water Xevo quadrupole timeof-flight mass spectrometer (MS; Manchester, UK), and its metabolic spectrum needs to be collected by an electrospray ionization (ESI) source. According to the preliminary experiment of the system determination, the spectral determination was carried out in the positive ion mode, and the optimum parameters are as follows: desolvation temperature of 350 °C, source temperature of 120 °C, cone voltage of 25 V and capillary voltage of 3.0 kV. Nitrogen with flow rate of 650 L/h and 50 L/h was used as nitrogen and conical gas respectively. The data acquisition rate of the instrument was adjusted to 0.2 s and the scanning interval delay is 0.02 s. The scanning range was 100-1000 m/z. In order to ensure the accuracy and reproducibility of the data, all analyses were done through the use of locking spray. Leucine-enkephalin was used as the positive ion model of acetonitrile (0.1% formic acid):H 2 O (0.1% formic acid) with a concentration of 400 pg/mL ([M + H] + = 556.2771). The Pareto mode was used to collect the data, and the frequency of phase-locked spray was set to 1 s. Before using the Mass Lynx 4.1 software to process the data, the average scanning of the phase-locked quality data was more than 10 times for correction.

DNA extraction and fungal identification
The mycelia of the two strains were inoculated into the flask containing 200 mL of PDB medium, and the mycelium biomass of about 100 mg was harvested after incubation on the shaker (160 rpm, 28 °C, 3 days). According to the research of Guo et al. (2001), the genomic DNA of endophytic fungi was isolated and extracted by the CTAB method.

Phylogenetic analysis
The BLAST algorithm was used to compare the corresponding ITS sequence and TEF-1α sequence of each strains with the NCBI (National Center for Biotechnology Information) data set on GenBank (NCBI; http:// www. ncbi. nlm. nih. gov). The DNAMAN program was used to perform multiple sequence alignments. Molecular evolutionary analysis and the phylogenetic tree construction were conducted using neighbor-joining method by MEGA version 11.0 (Kumar et al. 2008).

Results
To assay endophytic fungal metabolites with a reasonable elution time, isocratic elution of acetonitrile-water was performed as described in the experimental section. In our work, the metabolites of CHS 3 and CHS 2 (isolated from the stems of B. scorzonerifolium) with a running time of 17 min showed the same peaks as those of the SSd standards when eluted at 11.08 and 11.12 min, respectively. The metabolites of CHS 2 have the same cleavage fragments as the SSD standard at m/z 149.0204, 455.3502, 763.4695 (Fig. 1). The metabolites of CHS 3 regularly show several fragments at m/z 149. 0189, 437.3424, 455.3536, 601.4117 and 763.4784, which are the same as the cleavage fragments of SSd standard (Fig. 1). The amount of SSd produced by CHS 2 and CHS 3 were 2.17 and 2.40 µg/mL under the condition described in this study. After subculture, the two endophytic fungi still had a stable ability to produce SSd, and the strains were stored in degree cryogenic refrigerator at − 80 °C.
To further determine the phylogeny of strain CHS 2 and CHS 3 , the ITS1-5.8 S-ITS2 sequences and TEF-1α sequence of these isolates were amplified and sequenced (Fig. 2). Then the sequences were compared to corresponding sequences of referenced fungal taxa in the database.
Also, these fungi were identified by morphological characteristics (Fig. 3). CHS 2 grew rapidly in PDA medium, the colony was nearly round, the edge was neat, the colony was reddish in the middle, the edge was white, and the texture was loose and fluffy. According to the observation of mycelial microscope, the conidium was irregular and the conidia were chain-shaped. According to the morphological characteristics, CHS 2 was preliminarily identified as Fusarium sp. (Fig. 3). CHS 3 grew rapidly on PDA medium, the colony was round and uniform, and the hyphae were white, fluffy and well developed. Under microscope, the branches of conidiophores were irregular and distributed in groups. Thick-walled spores are formed in the peduncle of hyphae and cysts. According to the morphological characteristics, CHS 3 was preliminarily identified as Fusarium sp. (Fig. 3).
Based on ITS sequence and TEF-1α sequence, homologous sequence search with GenBank showed that the similarity between CHS 2 sequence and Fusarium oxysporum was 95% and 98% (GenBank accession numbers KC119203.1 and MT886217.1), and that between CHS 3 sequence and Fusarium acuminatum was 99% and 97% (GenBank accession numbers HM068320.1 and MF523228.1) ( Table 1). The phylogenetic relationship was established by comparing and branching the homologous nucleotide sequences of CHS 2 and CHS 3 in GenBank. According to the phylogenetic analysis, the isolates of CHS 2 and CHS 3 were classified as F. oxysporum and F. acuminatum,respectively (Figs. 4,5).
Identities were obtained by blasting sequences in NCBI

Discussion
Like host plants, endophytic fungi can also possess the natural capacity to produce bioactive secondary metabolites with the same or similar chemical structures (Lamshöft et al. 2008;Cui et al. 2012;Silvia et al. 2007). Therefore, endophytic fungi are considered as potential microbial resources to replace traditional medicinal plants to produce pharmacologically active natural compounds, and have very broad medicinal value and research prospects (Aly et al. 2011). Traditional medicinal plants are the top-priority for isolation and screening of endophytic fungi (Gómez and Luiz 2018). The discovery of SSd-producing endophytic fungi is valuable for industrial application. The production of SSd by endophytes CHS 2 and CHS 3 further supports the theory that through long-term symbiotic relationships between endophytic fungi and plants, endophytes may acquire genes from their host plants and could synthesize analogous or identical bioactive secondary metabolites to their hosts. This indicated that CHS 3 and CHS 2 could be substituted for B. scorzonerifolium to produce SSd in the fermentation industry. The genus Fusarium is widely distributed and is an important source of secondary metabolites, such as camptothecin and vincristine, so it has been widely concerned by people and has become the focus of research in the last few years (Kusari et al. 2009a, b). According to our concern, the genus Fusarium has not been found to have the ability to produce SSd before. In this paper, we first reported two SSd-producing endophytic fungi isolated from B. scorzonerifolium.
However, the SSd production by each of the two endophytes in axenic cultures is too low to match expectation. Because endophytes always interact with hosts and other endophytes, the biosynthesis ability of endophytic fungi in aseptic culture is very different from that in host plants. When observed metabolic production of the endophytic fungus isolated from Camptotheca acuminate, through the first to the seventh-generation subculture, Kusari et al. found that a sharp attenuation occurred in the production of camptothecin by this endophyte (Kusari et al. 2009a, b). They believe that the lack of host stimulation in aseptic cultures may be one of the reasons for the attenuation. It is highly probable that there might be metabolic communication between the endophytes and the host plants, and in vivo metabolic processes of endophytes are likely regulated by plants. Though the mechanisms by which endophytes interact with host plants are incompletely understood, we agreed with the theory that in the process   . The study of interspecific crosstalk between endophyte-endophyte and endophyte-host is worthy of further study. We have established endophytic-endophytic and endophytic-host co-culture models, and are currently studying the ways in which fungi and hosts interact to promote SSd production.

Conclusion
This is the first time that we have screened two endophytic fungi with stable genetic characters and the ability to produce SSd. CHS 2 and CHS 3 were identified as F. oxysporum and F. acuminatum respectively by ITS sequence and TEF-1α sequence analysis. All these efforts further prove the natural ability of endophytes to produce the same or

Conflict of interest
The authors have no relevant financial or non-financial interests to disclose.
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